Sulfonic stilbene compounds in the treatment of viral diseases

ABSTRACT

Sulfonic acid stilbenes block the infection of cells by HIV and these compounds can be used to prevent viral infection.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/463,582, filed Jun. 5,1995, U.S. Pat. No. 5,672,625, which is a divisional of application Ser.No. 08/248,851 filed May 25, 1994, now U.S. Pat. No. 5,494,932; which isa continuation of application Ser. No. 08/008,254, filed Jan. 25, 1993,now abandoned; which is a continuation of application Ser. No.07/829,132, filed Jan. 31, 1992, now abandoned; which is hereinincorporated by reference.

BACKGROUND OF THE INVENTION

A great deal of research is currently underway to develop treatments andcures for viral infections in humans and in animals. Notably theincidence of AIDS and ARC in humans is increasing at an alarming rate.The five year survival rate for those with AIDS is dispiriting and AIDSpatients, whose immune systems have been seriously impaired by theinfection, suffer from numerous opportunistic infections includingKaposi's sarcoma and Pneumocystis carninii pneumonia. No cure for AIDSis known and current treatments are largely without adequate proof ofefficacy and have numerous untoward side effects. Fear of the diseasehas resulted in social ostracism of and discrimination against thosehaving or suspected of having the disease.

Retroviruses are a class of ribonucleic acid (RNA) viruses thatreplicate by using reverse transcriptase to form a strand ofcomplementary DNA (cDNA) from which a double stranded, proviral DNA isproduced. This proviral DNA is then incorporated into the chromasomalDNA of the host cell making possible viral replication by transcriptionof this integrated DNA and translation of viral messenger RNA intoproteins; assembly of new viral RNA into a protein core and release fromthe cell results in the formation of infectious virus progeny.

Many of the known retroviruses are oncogenic or tumor causing. Indeedthe first two human retroviruses discovered, denoted human T-cellleukemia virus I and II or HTLV-I and II, were found to cause rareleukemias in humans after infection of T-lymphocytes. The third suchhuman virus to be discovered, HTLV-III, now referred to as HIV, wasfound to cause cell death after infection of T-lymphocytes and has beenidentified as the causative agent of acquired immune deficiency syndrome(AIDS) and AIDS related complex (ARC).

The envelope protein of HIV is a 160 kDa glycoprotein. The protein iscleaved by a protease to give a 120 kDa external protein, gp 120, and atransmembrane glycoprotein, gp 41. The gp 120 protein contains the aminoacid sequence that recognizes the receptor on CD4-positive humanT-helper cells. Recently it was reported that the polysulfatedpolysaccharides dextran sulfate, carrageenans of sea algae, pentosanpolysulfate, and heparin are highly effective inhibitors of HIV-1replication in vitro. M. Ito, et al., (1987) Antiviral. Res. 7, 361-367.Baba et al., Antiviral Res. 9, 335-343 (1988). O. Yoshida (1988)Biochem. Pharmacol. 37, 2887-2981. R. Ueno and S. Kuno, (1987) Lancet i,1379. The presence of the sulfate groups on these molecules is requiredfor the antiviral activity. The mechanism of this activity has beenstudied by Baba et al., (1988) Proc. Natl. Acad. Sci. USA, 85,6132-6136.

Applicants have discovered that a class of sulfonated stilbenes thatbear sulfonic acid groups are active against HIV. Herpes Simplex Viruses(HSV) I and II as well cytomegalovirus (CMV) have functionally relatedglycoprotein coatings and viral infectivity can also be diminished oreliminated by the use of the sulfonated stilbenes of this invention.

SUMMARY OF THE INVENTION

Compounds of formula 1 ##STR1## wherein

R₁ and R₂ are each independently a H₂ N--, O₂ N--, S═C═N--,

N.tbd.C--, or CH₃ C(O)NH-- group,

B is a --CH═CH--(cis or trans), --C.tbd.C--, or --CH₂ --CH₂ -- group,and

M₁ and M₂ are each independently a hydrogen or a pharmaceuticallyacceptable cation are useful in the treatment of diseases caused byinfections of eneloped viruses.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutically acceptable cations are those cations that are notsubstantially toxic at the dosage administered to acheive the desiredeffect and do not independently possess significant pharmacologicalactivity. Illustratively, these salts include those of alkali metals, asfor example, sodium and potassium; alkaline earth metals, such ascalcium and magnesium; light metals of Group IIIA including aluminum;and organic primary, secondary and tertiary amines, as for example,trialkylamines, including triethylamine, procaine, dibenzylamine,1-ethenamine, N,N'-dibenzylethylenediamine, dihydroabiethylamine,N(lower)alkylpiperidine, and any other suitable amine. Sodium salts arepreferred.

The compounds of this invention are readily prepared by the ordinaryartisan. The compounds are sulfonated derivatives of stilbene and can beprepared several ways by well known aromatic electrophilic substitutiontechniques such as are taught in R. T. Morrison and R. N. Boyd, OrganicChemistry, 5th ed., Allyn and Bacon, Inc., Boston, 1987, chap. 14. Ingeneral, the most efficient way to make the compounds of formula 1 is tosulfonate the corresponding nonsulfonated compound of formula 1a##STR2## wherein B, R₁, and R₂ are as defined above for the compounds offormula 1 or are groups readily transformable into such substitutents.Also, in general, when it is desired to prepare compounds of formula 1or 1a wherein B is other than a --C═C--, the corresponding compoundwherein B is a --HC═CH-- is first prepared and subsequently thecarbon-carbon bond is transformed by well known techniques to producethe desired moiety.

The compounds of formula 1a wherein B is a --HC═CH-- can be prepared,for example, by subjecting an optionally substituted benzaldehyde to abenzoin condensation as shown in Scheme A to form an initialketo-alcohol adduct of formula 2 ##STR3## wherein the B, R₁, and R₂groups are as defined above for the compounds of formula 1 or are groupswhich can be subsequently converted to the groups defined above. For agood general review of the benzoin condensation reaction see Ide andBuck, Org. Reactions 4, 269-304 (1948). The formula 2 intermediate isthen reduced to form a stilbene derivative, i.e. a compound of formula1a wherein B is a --HC═CH-- group. The reduction can be carried out inany conventional manner such as by use of a dissolving metal reductiontechnique using, for example, a zinc amalgam and a dilute acid such asacetic acid. The reduction usually results in a mixture of stilbenes ofcis and trans geometric configuration relative to the carbon-carbondouble bond, with the trans configuration preferred. One isomer can beconverted to the other isomer in any manner generally used by thoseskilled in the art to convert geometric isomers relative to acarbon-carbon double bond. For example, a trans stilbene can beconverted into a cis stilbene by the action of UV light.

Another approach to preparing the stilbenes of this invention isillustrated in scheme B. This reaction scheme involves the reaction ofan aldehyde of formula 3 with a benzyl Grignard of formula 4 to form analcohol of formula 5, which upon dehydration yields the desiredstilbene. The substitutents B, R₁, and R₂ of the formula 3, 4, and 5compounds are as defined above for the compounds of formula 1 or aregroups which can be subsequently converted to such substituents.##STR4##

Another approach to the preparation of the stilbenes of formula la isillustrated in scheme C involves the reaction of the aldehyde of formula3 with an alpha-phenyl acetate of formula 6 to form an alcohol ester offormula 7 which upon dehydration gives the acrylate of formula 8 andwhich upon acid promoted decarboxylation gives the stilbene of formula1a. ##STR5##

Applicants prefer those compounds of formula 1 wherein B is a --CH₂--CH₂ group and more prefer those wherein B is a --CH═CH-- group,especially those of the trans configuration. Applicants also preferthose compounds of formula 1 wherein R₁ and R₂ are each independently aS═C═N-- or a CH₃ C(O)N-- group. Applicants further prefer thosecompounds of formula 1 wherein M₁ and M₂ are each independently ahydrogen or a sodium cation. Applicants prefer those compounds whereinthe R₁ and R₂ groups are in the para (or 4) position relative to thecarbon to which the B group is attached and wherein the sulfonyl groupsare in the γ ortho (or 2) position relative to the carbon atom to whichthe B group is attached. The preferred compounds of this invention are4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H₂ DIDS),4-acetamido-4'-isothiocyantostilbene-2,2'-disulfonic acid (SITS) andespecially 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

The sulfonated stilbenes can be used to prevent infection of cells withHIV and syncytium formation in cells with established HIV infections, oragainst other related viruses having gp120 surface protein as well theHerpes Simplex Viruses (HSV) I and II and the cytomegalovirus (CMV). Thesulfonated stilbenes can be used to treat AIDS and ARC and otherdiseases caused by the retrovirus HIV or other related viruses havinggp120 surface protein as well as diseases caused by the Herpes SimplexViruses (HSV) I and II and cytomegalovirus (CMV).

When evaluated in the human T cell line JM acutely infected with the GB8virus, DIDS, H₂ DIDS and SITS (Table I) suppressed virus-inducedmultinucleated giant cell formation (syncytia). The antiviral activityof these compounds was confirmed by the dose-dependent inhibition of p24viral core antigen (Table I) and by the absence of viral infectivity inthe cell-free supernatants (data not shown). We also examined theantiviral properties of these compounds in a second host-cell/virussystem, i.e., the highly CD4⁺ C8166 cells infected with the Haitianstrain of HIV-1, RF. As above, DIDS and H₂ DIDS blocked HIV-1 infection;however, SITS at 100 μg/ml was inactive as determined by the syncytialand p24 antigen assays (Table I). DIDS and H₂ DIDS also blocked thegrowth of RF in MT-4 cells at concentrations that were noncytotoxic asdetermined by the tetrazolium reduction assay method (Nakashima et al.,1989), whereas SITS only had weak antiviral activity (not shown). In allthree host cell/virus systems, the antiviral potency was consistentlyDIDS>H₂ DIDS>>SITS.

                  TABLE I    ______________________________________    ANTIVIRAL ACTIVITY OF THE STILBENE DISULFONIC ACID    ANALOGS DIDS, H.sub.2 DIDS AND SITS AGAINST INFECTION OF JM    CELLS WITH HIV-1 STRAINS GB8 AND RF                          Mean             Concentration                          Syncytail.sup.a                                    P24 Antigen.sup.b    Compound μM        Counts    % Virus Control    ______________________________________    Virus Control             --           101       100    DIDS     200          0        0.02    (4.8).sup.c             100          0        0.56    (33)             50           0        6.4     (83)             25           31       100     (100)             12           101      --      --             6            114      --      --    H.sub.2 DIDS             200          0        0       (2.5)             100          0        0.01    (59)             50           7        43      (>100)             25           71       100     --             12           97       100     --             6            107      --      --    SITS     200          0        1.6     (>100)             100          15       85      (>100)             50           63       100     (>100)             25           63       --      --             12           72       --      --             6            93    ______________________________________     .sup.a At day 4 p.i.     .sup.b Virus control = 7.35 × 10.sup.4 pg/ml at day 6 p.i.     .sup.c Values in parenthesis are for the RF strain of HIV1 in C8166 cells

The stilbene disulfonic acids blocked HIV-1 replication when thecompounds were added within 2 hours of virus adsorption, i.e., prior tovirus penetration, and the cells were cultured in the presence of theinhibitors. These compounds were next examined for antiviral activity atlater stages of the infection process. At a MOI of 0.01 infectiousunits/cell (GB8 virus), viral glycoproteins appear at the surface of JMcells by 24 hours post-infection (p.i.) and initiate fusion withneighboring CD4⁺ cells; the syncytial numbers show a good correlationwith the production of cell-free virus (Tyms et al., 1990). As is shownin Table 2, the addition of 100 μg/ml DIDS and H₂ DIDS at 24 hours p.i.completely suppressed fusion relative to virus control when scored at 52hours and 68 hours p.i. In fact, when DIDS was added as late as 43 hoursp.i. to cultures containing substantial numbers of syncytial, thesesyncytia were completely absent by 68 hours p.i., and had significantlyregressed in the H₂ DIDS treated cultures. SITS, however, had noanti-syncytial activity relative to virus control although p24 antigenwas slightly reduced, indicating a reduced antiviral effect. Therelative order of antiviral potency of the stilbene disulfonic acidsagainst established HIV-1 infections is consistent with that observed inacute infections, i.e., DIDS>H₂ DIDS>>SITS suggesting a common mode ofantiviral action at late and early stages of infection.

                  TABLE II    ______________________________________    EFFECT OF DIDS, H.sub.2 DIDS AND SITS ON ESTABLISHED    INFECTIONS OF HIV-1 (STRAIN GB8) IN JM CELLS               Time of Syncytia.sup.b   ng/ml               Addi-   at time of                                Synctia at                                        P24 Antigen    Compound   tion (h).sup.a                       addition 52 h.sup.b                                     68 h.sup.b                                          75 h    ______________________________________    DIDS (100 μg/ml)               0       0        0    0    Neg.sup.c                                               (0)               24      0        0    0    Neg  (0)               43      9        19   0    2.8  (2.4%)    H.sub.2 DIDS               0       0        0    0    Neg  (0)    (100 μg/ml)               24      0        0    0    2.4  (2%)               43      9        19   3    16.4 (14%)    SITS (100 μg/ml)               0       0        4    27   6.7  (7.4%)               24      0        35   77   21.7 (18.6%)               43      9        59   80   49.3 (42.3%)    Drug-free medium               43      9        53   85   116.2                                               (100%)    ______________________________________     .sup.a Time relates to hours p.i.     .sup.b Mean syncytia numer (n = 3) at the time of addition of compound.     During the period of incubation from 43 to 52 hours p.i. the mean number     of syncytia increased from 9 to 53 synctia per well in the drugfree     controls.     .sup.c Neg = no antigen detected.

Syncytial cell formation depends on the interaction of gp120, expressedin the membranes of infected cells, with the CD4 antigen on neighboringcells (Camerini and Seed, 1990). We utilized a cell cocultivation modelto examine the effect of the stilbene disulfonic acids on the gp120-CD4dependent fusion process. This system consists of mixing H9 cellschronically infected with HIV-1 RF (gp120⁺ cells) with uninfected C8166cells (CD4⁺ cells) as the fusion indicator. In this assay, cell fusionoccurred within 2 to 3 hours of mixing at 37° C. and substantialsyncytia were observed after a further 3 to 4 hours (FIG. 1, Panel A).Treatment of cells with 100 μg/ml DIDS at the time of cell mixingcompletely prevented cell fusion (Panel B) while H₂ DIDS only partiallyprevented syncytia (not shown). SITS at 200 μg/ml (Panel C) had noeffect while DIDS at this concentration totally protected the C8166cells for 5 days post-mixing (not shown). In parallel experiments, 10μg/ml of 500,000 MW dextran sulfate (Panel E), like DIDS, completelyinhibited relative to the nontreated virus control (Panel D); however,250 μg/ml heparin (Panel F) and 100 μg/ml of 8,000 MW dextran sulfate(not shown) were totally ineffective. These findings with the sulfatedpolysaccharides were consistent with a previous report (Montefiori etal., 1990). Thus, where heparin and 8,000 MW dextran sulfate wereineffective at blocking virus-induced cell fusion and death, DIDS and H₂DIDS showed siginificant antiviral activity.

In the next experiments, CD4⁺ C8166 cells were pretreated overnight witheither DIDS (100 μg/ml), SITS (100 μg/ml) or drug-free medium.Unabsorbed compounds were removed by repeated washings and chronicallyinfected H9 cells were then added. As is shown in FIGS. 2A and B,pretreatment with SITS and drug-free medium, respectively, failed toblock syncytia over the 6 hour observation period. In contrast, theDIDS-treated cells (FIG. 2C) remained syncytia-free. In contrast, noprotective effect was observed when cells were pretreated with heparinor with 500,000 MW dextran sulfate. Thus, cell washing prior to mixingwith the H9 cells oblated the protective effect observed above (FIG. 1,Panel E) when 500,000 MW dextran sulfate was continually present in theassay.

The amount of sulfated stilbene of formula 1 which is needed to preventsyncytium formation in HIV, HSV or CMV infected cells can be anyeffective amount. Experimentally, applicants have determined thatsulfonated stilbenes when employed at a concentration of 50-100 μg/mlresulted in complete inhibition of syncytium formation as well asreduced the presence of P24 antigen, an indicator of HIV viralreplication, to below 3.0×10². The amount of sulfonated stilbene offormula 1 to be administered in order to treat AIDS or ARC or otherdisease caused by HIV infection as well as diseases caused by HSV andCMV infection can vary widely according to the particular dosage unitemployed, the period of treatment, the age and sex of the patienttreated, the nature and extent of the disorder treated, and otherfactors well-known to those practicing the appropriate arts. Moreoversulfonated stilbenes of formula 1 can be used in conjunction with otheragents known to be useful in the treatment of retroviral diseases andagents known to be useful to treat the symptoms of and complicationsassociated with diseases and conditions caused by retroviruses. Theanti-virally effective amount of sulfonic acid stilbenes of formula 1 tobe administered will generally range from about 15 mg/kg to 500 mg/kg. Aunit dosage may contain from 25 to 500 mg of the sulfonic acidstilbenes, and can be taken one or more times per day. The sulfonatedstilbenes of formula 1 can be administered with a pharmaceutical carrierusing conventional dosage unit forms either orally or parenterally.

For oral administration sulfonated stilbenes of formula 1 can beformulated into solid or liquid preparations such as capsules, pills,tablets, troches, lozenges, melts, powders, solutions, suspensions, oremulsions. The solid unit dosage forms can be a capsule which can be ofthe ordinary hard- or soft-shelled gelatin type containing, for example,surfactants, lubricants, and inert fillers such as lactose, sucrose,calcium phosphate, and cornstarch. In another embodiment the compoundsof this invention can be tableted with conventional tablet bases such aslactose, sucrose, and cornstarch in combination with binders such asacacia, cornstarch, or gelatin, disintegrating agents intended to assistthe break-up and dissolution of the tablet following administration suchas potato starch, alginic acid, corn starch, and guar gum, lubricantsintended to improve the flow of tablet granulations and to prevent theadhesion of tablet material to the surfaces of the tablet dies andpunches, for example, talc, stearic acid, or magnesium, calcium, or zincstearate, dyes, coloring agents, and flavoring agents intended toenhance the aesthetic qualities of the tablets and make them moreacceptable to the patient. Suitable excipients for use in oral liquiddosage forms include diluents such as water and alcohols, for example,ethanol, benzyl alcohol, and the polyethylene alcohols, either with orwithout the addition of a pharmaceutically acceptably surfactant,suspending agent, or emulsifying agent.

The sulfonated stilbenes of formula 1 may also be administeredparenterally, that is, subcutaneously, intravenously, intramuscularly,or interperitoneally, as injectable dosages of the compound in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid or mixture of liquids such as water, saline,aqueous dextrose and related sugar solutions, an alcohol such asethanol, isopropanol, or hexadecyl alcohol, glycols such as propyleneglycol or polyethylene glycol, glycerol ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant such as a soap or adetergent, suspending agent such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagent and other pharmaceutical adjuvants. Illustrative of oils which canbe used in the parenteral formulations of this invention are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil,petrolatum, and mineral oil. Suitable fatty acids include oleic acid,stearic acid, and isostearic acid. Suitable fatty acid esters are, forexample, ethyl oleate and isopropyl myristate. Suitable soaps includefatty alkali metal, ammonium, and triethanolamine salts and suitabledetergents include cationic detergents, for example, dimethyl dialkylammonium halides, alkyl pyridinium halides, and alkylamines acetates;anionic detergents, for example, alkyl, aryl, and olefin sulfonates,alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates;nonionic detergents, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers; andamphoteric detergents, for example, alkyl-beta-aminopropionates, and2-alkylimidazoline quarternary ammonium salts, as well as mixtures. Theparenteral compositions of this invention will typically contain fromabout 0.5 to about 25% by weight of the sulfonated stilbene in solution.Preservatives and buffers may also be used advantageously. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain a non-ionic surfactant having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. The surfactant can be a single component having the aboveHLB or can be a mixture of two or more components having the desiredHLB. Illustrative of surfactants used in parenteral formulations are theclass of polyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

REFERENCE TO THE DRAWINGS

FIG. 1

The effect of the stilbene disulfonic acids and various sulfatedpolysaccharides on virus-induced cell fusion. In this assay, 10⁵ H9cells, chronically infected with the HIV-1 strain RF, were coculturedwith 10⁵ uninfected C8166 cells in the absence and presence of the testcompounds. Panel A: Positive control, no compound addition; Panel B: 100μg/ml DIDS; Panel C: 100 μg/ml SITS; Panel D: positive control; Panel E:10 μg/ml 500,000 MW dextran sulfate; Panel F: 250 μg/ml heparin.

FIG. 2

Pretreatment of CD4⁺ C8166 cells with DIDS blocks fusion withchronically infected H9 cells. In this experiment, C8166 cells werepretreated overight with various stilbene disulfonic acids or drug-freemedium (fusion control). The cells were washed, incubated withchronically infected H9 cells and syncytia were photographed at 6 hourspost-mixing. Pretreatment conditions were as follows: Panel A: 100 μg/mlSITS; Panel B: drug-free medium; and Panel C: 100 μg/ml DIDS.

EXAMPLES

The following examples illustrate various aspects of this invention. Thefollowing information on reagents, cell lines, virus strains and assaysapply to Examples 1-7.

EXPERIMENTAL PROCEDURES

Reagents

4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS),4,4'-diisothicyanatodihydrostilbene-2,2'-disulfonic acid (H₂ DIDS) and4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) werefrom Molecular Probes, Inc. (Eugene, Oreg.). These compounds were >95%pure by reverse-phase HPLC diode array analysis; the system employs aHewlett Packard HP1040 detector coupled to a model 130A separationsystem (Applied Biosystems, Inc.) with an Aquapore RP-300 (2.1×30 mm)reverse-phase (C-8) column.

CD4⁺ Cell Lines and HIV-1 Strains

The cell lines used were the semi-mature human lymphoblastoid leukemia Tcell line JM, the human T-lymphoblastoid C8166 cell line, the humancutaneous T cell lymphoma line H9 and, the human T cell transformed lineMT-4. Stocks of HIV-1 strains RF and GB8 (kindly provided by Dr. GrahamFarrar, Center for Applied Microbiological Research, Porton Dawn,England) were prepared from chronically infected H9 cells and acutelyinfected JM cells, respectively. Virus stocks consisted of cell-freegrowth medium (RPMI 1640 plus 10% fetal calf serum) containing freshlyshed virus. Virus titres were determined by a standard plaque reductionassay.

Virus Infection Assays

Virus stocks (strains RF or GB8) were diluted into growth mediumcontaining various concentrations of DIDS, H₂ DIDS or SITS. The requirednumber of cells were then immediately added and the virus allowed toabsorb for 2 hours at 37° C. to give a multiplicity of infection (MOI)of 0.001 infectious units/cell. The virus-infected cells were pelleted,washed three times in phosphate buffered saline (PBS), and resuspendedin fresh growth medium containing the appropriate concentrations ofstilbene disulfonic acids. Cells were then distributed into 24-wellculture plates (Falcon) and assayed as described below for virusinfection. Alternatively, cells were first infected with HIV-1 (strainsRF or GB8) for 2 hours at 37° C. at the above MOI. After extensivewashing, cells were replated into fresh growth medium containing variousconcentrations of stilbene disulfonic acids. In the above experiments,syncytia were scored between 2 and 4 days postinfection (p.i.) intriplicate using an Olympus CK2 inverted microscope. The culture fluidswere recovered between 3 and 5 days p.i., clarified by low speedcentrifugation (2,000 rpm) for 5 minutes and the level of HIV-1 p24 coreantigen in the supernatants was determined by p24 antigen ELISA(Coulter). The concentrations of stilbene disulfonic acids giving 50%inhibition of HIV-1 growth (IC₅₀) were determined from the dose-responsecurves by linear regression analysis.

In post-treatent experiments, cells were first infected with HIV-1strain RF or GB8 for 2 hours at 37° C. at a MOI of 0.01 and allowed togrow for either 24 or 43 hours before replating into fresh growth mediumcontaining 100 μg/ml of DIDS, H₂ DIDS or SITS. Syncytia were then scoredat 52 and 68 hours p.i. in triplicate, and p24 viral core antigen inculture fluids were assayed at 75 hours p.i. as described above.

Anti-Syncytial Assay

CD4⁺ uninfected C8166 cells were seeded into 24-well trays (2×105cells/well) that contained growth medium and various concentrations oftest compounds. H9 cells, chronically infected with the RF strain ofHIV-1, were washed and then added as a source of gp120⁺ cells at 10⁵cells/well except for the noncytopathic control: those wells receiveduninfected H9 cells. The trays were then incubated at 37° C. and thecocultures were observed between 1 and 6 hours p.i. Syncytia wererecorded on photographic film.

In pretreatment experiments, CD4⁺ C8166 cells were maintained inserum-free medium (RPMI 1640 plus human transferrin 50 μg/ml, bovineinsulin 20 μg/ml, bovine serum albumin 2 mg/ml) and were exposed tocompound by overnight incubation. Treated cells were washed twice infresh serum-free medium and then cocultured with chronically infected H9cells in serum-free medium. Otherwise, cell numbers, assay conditionsand scoring of syncytia were as described above.

Example 1 Antiviral Activity of DIDS, H₂ DIDS, and SITS Against HIV-1

Protocol: JM cells were infected with HIV-1 to give a multiplicity ofinfection of 0.001. Virus adsorption was for 2 hours at roomtemperature. The cells were washed and distributed into wells containingdifferent concentrations of test compounds and incubated. After 2 daysthe cells were observed and syncytia were counted. The cell-freesupernatant fluids were harvested at 4 and 6 days post infection andassayed for levels of p24 viral core antigen and after 6 days thesupernatants were also titrated to determine levels of infectivity.

    ______________________________________    Results:    ______________________________________           Mean    Com-   Syncytial    pound  Counts    p24 pg/ml  %     p24 pg/m.                                             %    & Conc.           2 Days p.i.                     4 days p.i.                                Control                                      6 days p.i.                                             Control    ______________________________________    Virus  101       7.35 × 10.sup.4                                100%  2.18 × 10.sup.6                                             100%    Control    SITS    100 μg/ml           0         1.14 × 10.sup.3                                1.6%   2.2 × 10.sup.4                                             1%    50     15        6.25 × 10.sup.4                                85%   2.45 × 10.sup.5                                             11.2%    25     63        1.53 × 10.sup.5                                100%  1.26 × 10.sup.6                                             58%    12.5   63        1.85 × 10.sup.5                                100%  1.51 × 10.sup.6                                             69%    6      72        --               --     --    3      93        --               --     --    DIDS    100 μg/ml           0         None detected                                0     4.87 × 10.sup.2                                             0.02%    50     0         1.3 × 10.sup.2                                0.18% 1.23 × 10.sup.4                                             0.56%    25     0         4.7 × 10.sup.3                                6.4%  1.21 × 10.sup.6                                             56%    12.5   31        1.1 × 10.sup.5                                >100% 2.44 × 10.sup.6                                             >100%    6      101       --         --    --     --    3      114       --         --    --     --    H.sub.2 DIDS    100 μg/ml           0         None detected                                0     6.48 × 10.sup.2                                             0.03%    50     0         7.0 × 10.sup.1                                0.01% 3.77 × 10.sup.2                                             0.02%    25     7         3.2 × 10.sup.4                                43%   2.66 × 10.sup.6                                             >100%    12.5   71        9.4 × 10.sup.4                                >100% 8.51 × 10.sup.6                                             >100%    6      97        --         --    --     --    3      107       --         --    --     --    ______________________________________    BACK TITRATION OF CELL-FREE SUPERNATANTS    AT 6 DAYS p.i. (SFUs)*    COMPOUND    μg/ml       *SFUs/ml    ______________________________________    DIDS        100            <10.sup.1 /ml                50             <10.sup.1 /ml                25             Not done                12.5           2.4 × 10.sup.3 /ml    SITS        100            <10.sup.1 /ml                50             <10.sup.1 /ml                25             1.9 × 10.sup.3 /ml                12.5           2.1 × 10.sup.3 /ml    SITS        100            <10.sup.1 /ml                50             4.8 × 10.sup.3                25             2.5 × 10.sup.3                12.5           4.9 × 10.sup.3    Virus Control                --             2.6 × 10.sup.3    ______________________________________     *Syncytia-forming unit; cellfree supernatants from virus infected cells     having received the indicated concentration of compounds were incubated     with fresh uninfected JM cells and syncytia were scored to determine the     number of infectious units/ml. These results were compared to the cellfre     supernatants from virusinfected control cells not having received drug     treatment.

Example 2 Treatment of HIV-1 Infected Cells with DIDS, SITS, and ddC

Protocol: JM cells were infected with HIV to give approximately 100syncytia after 3 days. Virus adsorption was at room temperature for 2hours. The cells were then washed thoroughly and distributed in wells ofa tissue culture plate at a concentration of 1×10⁵ cells/well. At 24hours post infection either DIDS (100 μg/ml), SITS (100 μg/ml), or ddC(5 μM) were added to wells having infected cells. As virus controls,some wells had no drug added. At 42 hours post infection other wellsreceived either ddC (5 μM), SITS (100 μg/ml), or DIDS (100, 50, 25,12.5, 6.25 μg/ml). Syncytia were scored at 3 and 4 days post-infectionand the supernatant fluids were harvested after 3 and 4 days for p24core antigen determination.

    ______________________________________    Results:    ______________________________________    COMPOUND          SYNCTYTIA/ p24 ANTIGEN    and Time of       WELL       pg/ml    Addition CONC.    3 DAYS p.i.                                 3 DAYS p.i.                                          % Control    ______________________________________    Virus Control             --       >100        4.7 × 10.sup.4                                          100    DIDS at  100 μg/ml                      0           6.1 × 10.sup.3                                          13    24 hr    DIDS at  100 μg/ml                      34         1.98 × 10.sup.4                                          42    42 hr    50 μg/ml                      >80        1.92 × 10.sup.4                                          41             25 μg/ml                      As VC      1.50 × 10.sup.4                                          32             12 μg/ml                      As VC      2.10 × 1O.sup.5                                          >100             6 μg/ml                      As VC      1.80 × 10.sup.4                                          38    SITS     100 μg/ml                      As VC      1.80 × 10.sup.4                                          38    24 hr    SITS     100 μg/ml                      As VC      7.00 × 10.sup.3                                          15    42 hr    ddC 24 hr             5 μM  65         9.00 × 10.sup.3                                          19    ddC 42 hr             5 μM  As VC      1.70 × 10.sup.4                                          36    ______________________________________                      MEAN                      SYNCTYTIA/ p24 Antigen                      WELL       pg/ml   %    COMPOUND CONC.    4 DAYS p.i.                                 4 DAYS p.i.                                         CONTROL    ______________________________________    Virus Control             --       >150       8.95 × 10.sup.4                                         100    DIDS     100 μg/ml                      20         1.58 × 10.sup.4                                         18    24 hr    DIDS     100 μg/ml                      70         2.09 × 10.sup.4                                         23    42 hr    50 μg/ml                      100        6.3 × 10.sup.4                                         70             25 μg/ml                      As VC      5.9 × 10.sup.4                                         66             12 μg/ml                      As VC      3.9 × 10.sup.5                                         44             6 μg/ml                      As VC      3.4 × 10.sup.4                                         38    SITS     100 μg/ml                      As VC      6.25 × 10.sup.4                                         70    24 hr    SITS     100 μg/ml                      As VC      9.5 × 10.sup.4                                         >100    42 hr    ddC 24 hr             5 μM  51         3.1 × 10.sup.3                                         3.5    ddC 24 hr             5 μM  As VC      6.7 × 10.sup.4                                         75    ______________________________________

Example 3 Anti-HIV Activity of DID, SITS, H₂ DIDS

Protocol; H9 cells chronically infected with RF strain of HIV-1 anduninfected C8166 cells were mixed in the presence of either DIDS, SITS,H₂ DIDS, dextran sulfate, or heparin (100 μg/ml). the cells wereobserved after 2, 3-1/2 and 5-1/2 hours and the amount of cell fusionquantified.

    ______________________________________    Results:    ______________________________________                         Syncytia                         after    after  after    COMPOUND    CONC.    2 hrs    31/2 hrs                                         51/2 hrs    ______________________________________    Virus Control                --       ++       +++    +++    DIDS        100 μg/ml                         0        0      0    SITS        100 μg/ml                         ++       ++     ++    H.sub.2 DIDS                100 μg/ml                         0        0      0    Heparin     100 μg/ml                         ++       +++    +++    Dextran     100 μg/ml                         0/+      0      0    Sulfate    ______________________________________

Example 4 Inhibition of HIV Induced Cell Fusion After Pretreatment ofUninfected Cells with DIDS, SITS and H₂ DIDS

Protocol: C8166 cells were pretreated for 2.5 hours with either DIDS,SITS or H₂ DIDS (at 500 and 250 μg/ml). The cells were then washed threetimes and mixed with H9 cells chronically infected with HIV-1. Cellfusion was quantified after 4 and 24 hours.

    ______________________________________    Results:    ______________________________________                           Syncytial Scores    COMPOUND   CONC.       4 hrs p.i.                                    24 hrs p.i.    ______________________________________    Virus Control               --          ++++     ++++    SITS       500 μg/ml                           +++/++++ ++++               250 μg/ml                           +++/++++ ++++    DIDS       500 μg/ml                           +        +++               250 μg/ml                           ++/+++   +++    H.sub.2 DIDS               500 μg/ml                           +        +++               250 μg/ml                           ++/+++   ++++    ______________________________________

Example 5 Anti-HIV Activity of DIDS, SITS and H₂ DIDS

Protocol: H9 cells chronically inected with HIV-1 and uninfected C8166cells were mixed in the presence of either DIDS, SITS, H₂ DIDS, dextransulfate, heparin (at 100 μg/ml), OKT4 or OKT4A (at a 1/50 dilution). Thecells were observed after 4 hours and the amount of cell fusionquantitied. OKT4 and OKT4A are monoclonal antibodies that bined the CD4cell surface antigen.

    ______________________________________    Results:                             Syncytia    COMPOUND        CONC.    after 4 hrs    ______________________________________    Virus Control   --       +++    DIDS            100 μg/ml                             0/+    H2DIDS          100 μg/ml                             0/+    SlTS            100 μg/ml                             +++    Heparin         100 μg/ml                             +++    Dextran sulfate 100 μg/ml                             ++    (8,000)    Dextran sulfate 100 μg/ml                             0    (500,000)    OKT4            1/50     +++    OKT4A           1/50     0    ______________________________________

Example 6 Effects of DIDS, H₂ DIDS and SITS on Established Infections ofHIV-1

Protocol: JM cells were infected with HIV at a multiplicity of infectionof 0.00025 syncytial forming units per cell. Virus adsorption was atroom temperature for one hour. The cells were then washed three timesand distributed in wells of a tissue culture plate at a concentration of2×10⁵ cells/well. Either DIDS, SITS or H₂ DIDS (all at 100 μg/ml) wereadded to wells at 0, 24, 43 and 51 hours post-infection. As viruscontrols some wells had no drug added. Syncytia were scored at 43, 52and 68 hours post-infection. the cell-free supernatant fluids of thevirus control wells were harvested after 33, 43, 51, 68 and 75 hourspost-infection. All other wells were harvested 75 hours post-infection.The supernatant fluid was then assayed for p24 viral core antigen.

    ______________________________________    Results:    ______________________________________    Time course of syncytial formation and p24 Antigen    Production    Time          Mean Syncytia                             P24 pg/ml    ______________________________________    33 hours      0          neg (<10.sup.3)    43 hours      9          neg    51 hours      53         2383    68 hours      85         13678    75 hours      0          116196    ______________________________________    P24 Antigen (75 hours post-infection) (pg/ml)                Time of Addition (hours)    Compound    0        24       43     51    ______________________________________    DIDS        Neg      Neg      2793   49050    100 μg/ml                >1%      <1%      2.4%   42%    SITS        8680     21669    49245  53830    100 μg/ml                7.4%     18.6%    42.3%  47.2%    H.sub.2 DIDS                Neg      2363     16412  42020    100 μg/ml                <1%      2%       14%    36%    ______________________________________    Syncytial Scores              Time              of Addition     Mean Syncytia                                       Mean Syncytia    COMPOUND  (hours)         51 hrs   68 hrs    ______________________________________    DIDS       0h    (0)*     0        0              24h    (0)      0        0              43h    (9)      19       0              51h    (53)     --       25    SITS       0h    (0)      4        27              24h    (0)      35       77              43h    (9)      59       As VC              51h    (53)     --       As VC    H.sub.2 DIDS               0h    (0)      0        0              24h    (0)      0        0              43h    (9)      19       3              51h    (53)     --       15    Virus Control              --              53       85    ______________________________________     *Mean Syncytia at time of addition of compound

Example 7 Effect of Sulfonated Stilbenes on HSV and CMV Growth

Protocol: Plaque reduction assay. Treatment after adsorption duringincubation.

    ______________________________________    Results-:    ______________________________________    HCMV                         PLAQUE    COMPOUND  CONC. μg/ml                         COUNTS         MEAN % VC    ______________________________________    DIDS      1000 μg 0     0   0        0              100        3     4   3        10%              10         16    12  11       43%              1          15    14  --       49%    Virus Control              --         34    30  31  27   100%    ______________________________________    HCMV    COMPOUND  CONC.     COUNTS          MEAN % VC    ______________________________________    Gancyclovir              1000 μg/ml                        0     0    0        0              10        3     2    7        10%              1         6     13   1        22%              0.1       34    39   --       >100%    Virus Control              --        30    31   27  34   100%    ______________________________________    HSV 2    COMPOUND  CONC.     COUNTS          MEAN % VC    ______________________________________    DIDS      100 μg/ml                        0     0    0        0              10        25    25   25       44%              1         31    33   39       61%              0.1       37    40   --       68%    Virus Control              --        65    51   58  51   100%    H.sub.2 DIDS              100 μg/ml                        0     0    0        0              10        26    19   21       43%              1         23    31   24       46%              0.1       35    35   --       63%    Virus Control              --        27    20   24  25   100%    ______________________________________    HSV2    COMPOUND  CONC.     COUNTS          MEAN % VC    ______________________________________    SITS      100 μg/ml                        16    13   --       63%              10        25    25   24       100%              1         28    25   23       100%              0.1       34    26            >100%    Virus Control              --        27    20   24  25   100%    Acyclovir 100 μg/ml                        0     0    0        0              10        0     1    0        0              1         31    31   29       54%              0.1       44    34   --       70%    Virus Control              --        65    51   58  51   100%    ______________________________________

What is claimed is:
 1. A method of treating an HIV viral infection in apatient in need thereof comprising administering to the patient anantivirally effective amount of a compound of the formula ##STR6##wherein R₁ and R₂ are each independently a H₂ N--, O₂ N--, S═C═N--,N.tbd.C--, or CH₃ C(O)NH-- group,B is a --HC═CH-- (cis or trans),--C.tbd.C-- or --CH₂ --CH₂ -- group, and M₁ and M₂ are eachindependently a hydrogen or pharmaceutically acceptable cation.
 2. Amethod of claim 1 wherein B is a --HC═CH-- group.
 3. A method of claim 1wherein R₁ and R₂ are independently a S═C═N-- or CH₃ (CO)N-- group.
 4. Amethod of claim 1 wherein the sulfonyl groups are at the ortho position.5. A method of claim 1 wherein M₁ and M₂ are each independently ahydrogen or a sodium cation.
 6. A method claim 1 wherein the compound is4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid.
 7. A method of claim1 wherein the compound is4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid.
 8. A methodof claim 1 wherein the compound is4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid.